The LysR family transcriptional regulator ORF-L16 regulates spinosad biosynthesis in Saccharopolyspora spinosa

Abstract

Spinosad, a potent broad-spectrum bioinsecticide produced by Saccharopolyspora spinosa, has significant market potential. Despite its effectiveness, the regulatory mechanisms of spinosad biosynthesis remain unclear. Our investigation identified the crucial role of the LysR family transcriptional regulator ORF-L16, located upstream of spinosad biosynthetic genes, in spinosad biosynthesis. Through reverse transcription PCR (RT-PCR) and 5'-rapid amplification of cDNA ends (5'-Race), we unveiled that the spinosad biosynthetic gene cluster (BGC) contains six transcription units and seven promoters. Electrophoretic mobility shift assays (EMSAs) demonstrated that ORF-L16 bound to seven promoters within the spinosad BGC, indicating its involvement in regulating spinosad biosynthesis. Notably, deletion of ORF-L16 led to a drastic reduction in spinosad production from 1818.73 mg/L to 1.69 mg/L, accompanied by decreased transcription levels of spinosad biosynthetic genes, confirming its positive regulatory function. Additionally, isothermal titration calorimetry (ITC) and EMSA confirmed that spinosyn A, the main product of the spinosad BGC, served as an effector of ORF-L16. Specifically, it decreased the binding affinity between ORF-L16 and spinosad BGC promoters, thus exerting negative feedback regulation on spinosad biosynthesis. This research enhances our comprehension of spinosad biosynthesis regulation and lays the groundwork for future investigations on transcriptional regulators in S. spinosa.

Keywords: LysR family transcriptional regulator; Regulation; Saccharopolyspora spinosa; Spinosad; Spinosyn A.

Fig. 1

Analysis of the upstream and downstream genes of spinosad biosynthetic genes. (A) BLAST analysis of the upstream and downstream genes of spinosad biosynthetic genes. (B) Multiple amino acid sequence alignment of the DBDs of the ORF-L16 and LysR family transcriptional regulators with complete crystal structures. CbnR is from Burkholderiales [30], BenM is from Acinetobacter [37], YfbA is from Yersinia pestis, and DarR is from Aliivibrio fischeri [38].

Fig. 2

The transcription units and TSSs of the spinosad BGC. (A) The transcription units of the spinosad BGC were analyzed by RT-PCR. (B) Gel electrophoresis of RT-PCR products. (C) 5′-Race was used to characterize the TSSs of the six transcription units within the spinosad BGC, and the −35 region and −10 region of the seven promoters as well as the interval between these two regions were inferred.

Fig. 3

EMSAs of spinosad BGC promoters and ORF-L16. (A) SDS-PAGE electrophoresis of the ORF-L16 protein. The (B) spnQ promoter, (C) spnP1 promoter, (D) spnP2 promoter, (E) spnI promoter, (F) spnJ promoter, (G) spnF promoter, and (H) spnG promoter interacted with the ORF-L16 protein in a concentration-dependent and specific manner. Hollow arrows indicated the free promoter, solid arrows indicated the complex of the promoter and protein. The EMSA results are representative examples of three independent experiments.

Fig. 4

Effects of ORF-L16 deletion and overexpression on spinosad production. (A) Spinosad productions of engineered strains with deletion and overexpression ORF-L16 and the control strain WHU1123. Error bars are standard deviations from three independent experiments, and P values were tested by Student’s t-test. ****, P ≤ 0.0001. (B) Logarithmic transformation of the transcriptional level ratio between the ΔORF-L16 and WHU1123 strains for spinosad biosynthetic genes. RNA sequencing was conducted in three independent experiments, and the P values are shown in Table S3.

Fig. 5

The interaction between spinosyn A and ORF-L16. (A) AutoDock Vina was used to simulate the molecular docking of the predicted ORF-L16 protein structure and spinosyn A. The NH of tryptophan indole group at position 196 in ORF-L16 formed a hydrogen bond with the oxygen atom in the 2′-methoxy group of spinosyn A rhamnose, with a distance of 2.8 Å. (B) Effect of spinosyn A on interaction between ORF-L16 and the spnQ promoter. Hollow arrows indicated the free promoter, solid arrows indicated the complex of the promoter and protein. The EMSA result is the representative example of three independent experiments. (C) ITC curve of the interaction of ORF-L16 with spinosyn A at 30 °C. The integrated injection heats derived from the titrations, corrected for control dilution heat. (D) The solid line is the best-fit curve and was used to derive the binding parameter.